The number of proteins produced by the human genome likely numbers in the hundreds of thousands. However, recent evidence indicates that the human genome contains only 30,000 to 45,000 different genes. Clearly, each gene is producing multiple proteins.
Alternative splicing of primary RNA transcripts is a major mechanism for increasing production of proteins from the human genome. It is known that 30% to 60% of genes undergo alternative splicing to produce messenger RNA (mRNA). Modrek B et al. Nat. Genet. 30, 13-19 (2002). These alternatively spliced mRNA are translated into alternative splice form proteins that contain amino acid sequences different than the corresponding protein produced by normally spliced mRNA.
Alternative splice form proteins are often expressed in a tissue-specific manner, or under certain physiologic or disease states. Modrek B et al., Nucl. Acids Res. 29, 2850-2859 (2001). Consequently, certain alternatively spliced mRNA are present in a limited number of cells in a subject suffering from a given disease or condition. For example, it is known that many types of cancer cells produce alternative splice forms which are not found in normal cells from the same subject. Cancer-associated genes such as CD44 (Rodriguez C et al., Int. J. Cancer 64, 347-354, 1995), estrogen receptor (Castles C G et al., Cancer Res. 53, 5934-5939, 1993), FGF receptor (Luqmani Y A et al., Int. J. Cancer 64, 274-279, 1995), DNA polymerase (Bhattacharyya N et al., DNA Cell Biol. 18, 549-554, 1999), cathepsin B (Gong Q et al., DNA Cell Biol. 12, 299-309, 1993), FHIT (Panagopoulos I. et al., Cancer Res. 56, 4871-4875, 1996), BRCA1 (Thakur S et al., Mol. Cell Biol. 17, 444-452, 1997) and BRCA2 (Bieche I et al., Cancer Res. 59, 2546-2550, 1999), produce alternatively spliced mRNA that are specifically expressed in cancerous tissues. Other disease states in which alternative splice forms are specifically produced in certain tissues include diabetes, Alzhiemer's disease and systemic lupus erythematosus (SLE).
Drugs that target proteins specific to cancerous or other disease tissue have proven efficacious in the appropriate patient population. For example, successful treatment of breast cancer has been reported for drugs which target the estrogen receptor (Jordan C, Clin. Ther. 24 Suppl A, A3-16, 2002) or the HER-2 receptor (Thomssen C, Anticancer Drugs 12 Suppl 4, S19-S25, 2001; Yip Y L et al., Cancer Immunol. Immunother. 50; 569-587, 2002). The genetic alterations present in tumor-specific proteins, such as mutations in p53, BRCA 1 and BRCA2, provide another source of targets. Thus, the proteins produced from alternatively spliced mRNA produced specifically in cancers or other disease states are also attractive therapeutic targets.
However, proteins produced from alternatively spliced mRNA have not been widely exploited as therapeutic targets. The major impediment to using such proteins as therapeutic targets has been the incidental or tedious nature by which alternatively spliced mRNA are found. Present methodologies are limited to either cDNA cloning (which is highly labor intensive) or RT/PCR (which focuses only on known portions of genes). In addition, most cloning- and RT/PCR-based methods are highly biased, as they require prior knowledge of the alternatively spliced mRNA sequence.
An unbiased procedure for discovery of alternatively spliced mRNA has been reported in U.S. Pat. No. 6,251,590 of Schweighoffer et al. However, the Schweighoffer et al. method identifies only the region in the alternatively spliced mRNA that is different from the normally spliced mRNA. The cDNA corresponding to both the normal and alternatively spliced mRNA must be separately cloned in order to pinpoint the alternatively spliced region in the context of the full-length molecule. The sequencing of multiple cDNA clones is also required to determine the prevalence of a given alternatively spliced mRNA. The Schweighoffer et al. method thus required a substantial investment of both time and resources in order to identify alternatively spliced molecules.
Thus, an unbiased method of rapidly and easily identifying alternatively spliced RNA in biological sample is needed, in which both the full-length normal and alternatively spliced mRNA are simultaneously isolated for comparison. Ideally, such a method would not rely on multiple cloning and sequencing steps for determining the identity and relative abundance of alternative splice forms in a given sample.